STRUCTURE/FUNCTION STUDIES ON PLASMINOGEN AND PLASMIN

Summary

Principal Investigator: Francis Castellino
Abstract: The long range objective of our research is to understand the structure-function relationships of proteins that are responsible for blood clot dissolution. In this application we are focusing on the protein chemistry, enzymology, immunochemistry, spectroscopic properties and molecular biology of human plasminogen, tissue plasminogen activator, urokinase, streptokinase and fibrin(ogen). The above proteins have been proposed to contain regions that can be classified as domains,which have residing within them the different functions of the proteins. The central hypothesis of this proposal is that specific properties of these molecules, such as fibrin binding, epsilon-aminocaproic acid binding, inhibitor binding, anion binding, protease sites that regulate function, etc., can be integrally removed and/or inserted into other proteins by recombinant DNA methodology to provide variant proteins with strategically altered properties. We first propose to examine, by calorimetric, immunologic and NMR methodology, the degree of independence of the domain regions (e.g., kringles, growth factor, finger) in the recombinant proteins and in a variety of mutant proteins. The conformational determinants and individual ligand binding properties of these domains will be investigated through a powerful combination of scanning and titration calorimetry, circular dichroism, high-field NMR, structural predictive techniques, synthesis of functional peptides and evaluation of the functional consequences of interaction of the proteins with antibodies to these synthetic peptides. In order to assess some aspects of structure-function relationships in these proteins, advantage will be taken of strategically-generated variant forms of the proteins, obtained through oligonucleotide-directed mutagenesis of their respective cDNAs, which lack, or have inserted within them, selected regions. Identification of important specific amino acids residues of the relevant proteins that are involved in their structural stabilities and interactions of interest will be undertaken by investigating the properties of synthetic peptides, and their corresponding antibodies, lacking or having inserted into them, specific amino acid alterations. The study will also include investigations on proteins and protein fragments with these same specific amino acid changes. Execution of this strategy will allow an increased understanding of the regions of the proteins that are responsible for their functions, and will provide the opportunity to generate proteins with engineered functions that will enhance or diminish their abilities to contribute to the reactions that lead to blood clot dissolution. Important new thrombolytic agents, and/or strategies for application of thrombolytic therapy to human vascular disease, is expected to directly or indirectly result from the basic knowledge provided by this work.
Funding Period: 1975-02-01 - 2000-08-31
more information: NIH RePORT

Top Publications

  1. pmc The β-domain of cluster 2b streptokinase is a major determinant for the regulation of its plasminogen activation activity by cellular plasminogen receptors
    Yueling Zhang
    W M Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA Institute of Animal Sciences, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
    Biochem Biophys Res Commun 444:595-8. 2014
  2. ncbi Solution structure and functional characterization of human plasminogen kringle 5
    Marcos D Battistel
    Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA
    Biochemistry 48:10208-19. 2009
  3. pmc Early platelet dysfunction: an unrecognized role in the acute coagulopathy of trauma
    Max V Wohlauer
    Department of Surgery, University of Colorado Denver, Denver, CO, USA
    J Am Coll Surg 214:739-46. 2012
  4. pmc Characterization of streptokinases from group A Streptococci reveals a strong functional relationship that supports the coinheritance of plasminogen-binding M protein and cluster 2b streptokinase
    Yueling Zhang
    W M Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Biol Chem 287:42093-103. 2012
  5. pmc Bacterial plasminogen receptors utilize host plasminogen system for effective invasion and dissemination
    Sarbani Bhattacharya
    W M Keck Center for Transgene Research, University of Notre Dame, Notre Dame, IN 46556, USA
    J Biomed Biotechnol 2012:482096. 2012
  6. pmc A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D Streptococcal pyogenes strain influences virulence-associated genes
    Zhong Liang
    WM Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Biol Chem 288:6561-73. 2013
  7. pmc Functional differences between Streptococcus pyogenes cluster 1 and cluster 2b streptokinases are determined by their β-domains
    Yueling Zhang
    W M Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
    FEBS Lett 587:1304-9. 2013
  8. pmc A key role for the urokinase plasminogen activator (uPA) in invasive Group A streptococcal infection
    Martina L Sanderson-Smith
    Ilawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia
    PLoS Pathog 9:e1003469. 2013
  9. pmc Complement-mediated opsonization of invasive group A Streptococcus pyogenes strain AP53 is regulated by the bacterial two-component cluster of virulence responder/sensor (CovRS) system
    Garima Agrahari
    From the W M Keck Center for Transgene Research and
    J Biol Chem 288:27494-504. 2013
  10. pmc Monoclonal antibodies detect receptor-induced binding sites in Glu-plasminogen
    Jaena Han
    Departments of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA
    Blood 118:1653-62. 2011

Scientific Experts

  • Lindsey Miles
  • Jennifer W Mitchell
  • Francis J Castellino
  • Victoria A Ploplis
  • Yueling Zhang
  • Zhong Liang
  • Mary Prorok
  • Min Wang
  • Garima Agrahari
  • Takayuki Iwaki
  • Martina L Sanderson-Smith
  • Mariana Figuera-Losada
  • James H Geiger
  • Jeffrey A Mayfield
  • Kristofor Glinton
  • Rashna D Balsara
  • Max V Wohlauer
  • Sarbani Bhattacharya
  • Mark J Walker
  • Marie Ranson
  • Jaena Han
  • Robert J Parmer
  • Marcos D Battistel
  • H Xu
  • Ana Gutierrez-Fernandez
  • Qihua Fu
  • F J Castellino
  • V A Ploplis
  • Sara E Cnudde
  • D Cruz-Topete
  • Deborah Donahue
  • Diane Ly
  • Victor Nizet
  • Vishwanatha Chandrahas
  • Andrew Hollands
  • Deborah L Donahue
  • Hsing Tse Hsueh
  • Jeffrey Harr
  • Victoria Ploplis
  • Ernest E Moore
  • Mark Walsh
  • Angela Sauaia
  • Scott Thomas
  • Christopher C Silliman
  • Ed Evans
  • Yun Gong
  • Jian Ming Yang
  • Kee Hwan Kim
  • Merce Jardi
  • Jordi Felez
  • Nagyung Baik
  • Gye Won Han
  • Jaroslav Zajicek
  • Seong Soo A An
  • M J Sandoval-Cooper
  • Neill A Gingles
  • D L Donahue
  • Miguel Llinas
  • Alexander Grishaev
  • H Menchen
  • Hongdong Bai
  • F Noria
  • Sara Cnudde
  • Mayra J Sandoval-Cooper
  • Markus Brechmann
  • T Iwaki

Detail Information

Publications23

  1. pmc The β-domain of cluster 2b streptokinase is a major determinant for the regulation of its plasminogen activation activity by cellular plasminogen receptors
    Yueling Zhang
    W M Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA Institute of Animal Sciences, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
    Biochem Biophys Res Commun 444:595-8. 2014
    ....
  2. ncbi Solution structure and functional characterization of human plasminogen kringle 5
    Marcos D Battistel
    Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA
    Biochemistry 48:10208-19. 2009
    ..Combined with previous studies, the observation of OA binding expands the range of ligands that interact with kringle 5 while it widens the scope of potential biological functions for kringle domains...
  3. pmc Early platelet dysfunction: an unrecognized role in the acute coagulopathy of trauma
    Max V Wohlauer
    Department of Surgery, University of Colorado Denver, Denver, CO, USA
    J Am Coll Surg 214:739-46. 2012
    ..Although the current debate on acute traumatic coagulopathy has focused on the consumption or inhibition of coagulation factors, the question of early platelet dysfunction in this setting remains unclear...
  4. pmc Characterization of streptokinases from group A Streptococci reveals a strong functional relationship that supports the coinheritance of plasminogen-binding M protein and cluster 2b streptokinase
    Yueling Zhang
    W M Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Biol Chem 287:42093-103. 2012
    ....
  5. pmc Bacterial plasminogen receptors utilize host plasminogen system for effective invasion and dissemination
    Sarbani Bhattacharya
    W M Keck Center for Transgene Research, University of Notre Dame, Notre Dame, IN 46556, USA
    J Biomed Biotechnol 2012:482096. 2012
    ..The goal of this paper is to highlight mechanisms whereby pathogenic bacteria, by engaging surface receptors, utilize and exploit the host plasminogen and fibrinolytic system for the successful dissemination within the host...
  6. pmc A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D Streptococcal pyogenes strain influences virulence-associated genes
    Zhong Liang
    WM Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Biol Chem 288:6561-73. 2013
    ..These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase...
  7. pmc Functional differences between Streptococcus pyogenes cluster 1 and cluster 2b streptokinases are determined by their β-domains
    Yueling Zhang
    W M Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
    FEBS Lett 587:1304-9. 2013
    ....
  8. pmc A key role for the urokinase plasminogen activator (uPA) in invasive Group A streptococcal infection
    Martina L Sanderson-Smith
    Ilawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia
    PLoS Pathog 9:e1003469. 2013
    ..These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections. ..
  9. pmc Complement-mediated opsonization of invasive group A Streptococcus pyogenes strain AP53 is regulated by the bacterial two-component cluster of virulence responder/sensor (CovRS) system
    Garima Agrahari
    From the W M Keck Center for Transgene Research and
    J Biol Chem 288:27494-504. 2013
    ....
  10. pmc Monoclonal antibodies detect receptor-induced binding sites in Glu-plasminogen
    Jaena Han
    Departments of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA
    Blood 118:1653-62. 2011
    ....
  11. pmc NMR backbone dynamics of VEK-30 bound to the human plasminogen kringle 2 domain
    Min Wang
    W M Keck Center for Transgene and the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
    Biophys J 99:302-12. 2010
    ....
  12. pmc Effects on human plasminogen conformation and activation rate caused by interaction with VEK-30, a peptide derived from the group A streptococcal M-like protein (PAM)
    Mariana Figuera-Losada
    W M Keck Center for Transgene Research, University of Notre Dame, Notre Dame, IN 46556, USA
    Biochim Biophys Acta 1804:1342-9. 2010
    ..These results are in contrast with the major conformational changes elicited by small-molecule activators of Pg, and point towards a novel mechanism of Pg activation that may underlie group A streptococcal (GAS) virulence...
  13. ncbi A fibrinogen deficiency accelerates the initiation of LDL cholesterol-driven atherosclerosis via thrombin generation and platelet activation in genetically predisposed mice
    Takayuki Iwaki
    W M Keck Center for Transgene Research, 230 Raclin Carmichael Hall, University of Notre Dame, Notre Dame, IN 46556, USA
    Blood 107:3883-91. 2006
    ....
  14. pmc Plasminogen inhibits TNFalpha-induced apoptosis in monocytes
    Jennifer W Mitchell
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    Blood 107:4383-90. 2006
    ..Because apoptosis of monocytes plays a key role in inflammation and atherosclerosis, these results provide insight into a novel role of plasminogen in these processes...
  15. ncbi X-ray crystallographic structure of the angiogenesis inhibitor, angiostatin, bound to a peptide from the group A streptococcal surface protein PAM
    Sara E Cnudde
    Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA
    Biochemistry 45:11052-60. 2006
    ..This dimerization, which exists only in the crystal structure, is consistent with the parallel coiled-coil full-length PAM dimer expected from sequence similarities and homology modeling...
  16. ncbi Delayed inflammatory responses to endotoxin in fibrinogen-deficient mice
    D Cruz-Topete
    WM Keck Center for Transgene Research and the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
    J Pathol 210:325-33. 2006
    ..These results suggest that fibrin(ogen) plays a role as an early mediator in the cross-talk between coagulation and inflammation...
  17. ncbi The lack of binding of VEK-30, an internal peptide from the group A streptococcal M-like protein, PAM, to murine plasminogen is due to two amino acid replacements in the plasminogen kringle-2 domain
    Qihua Fu
    W M Keck Center for Transgene Research and the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
    J Biol Chem 283:1580-7. 2008
    ..These mechanisms for accumulation of protease activity on GAS likely contribute to the virulence of PAM(+)-GAS strains and identify targets for new therapeutic interventions...
  18. pmc The hemostasis system in murine atherosclerosis
    Takayuki Iwaki
    W M Keck Center for Transgene Research, Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
    Curr Drug Targets 9:229-38. 2008
    ..In order to summarize recent progress in this area, this review is focused on mice lacking individual hemostasis genes and their contributions to steps of the atherosclerotic process...
  19. ncbi Severe deficiency of coagulation Factor VII results in spontaneous cardiac fibrosis in mice
    H Xu
    W M Keck Center for Transgene Research and the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
    J Pathol 217:362-71. 2009
    ....
  20. pmc Solution structure of the complex of VEK-30 and plasminogen kringle 2
    Min Wang
    W M Keck Center for Transgene Research, 230 Raclin Carmichael Hall, University of Notre Dame, Notre Dame, IN 46556, USA
    J Struct Biol 169:349-59. 2010
    ..Structural analysis and kringle sequence alignments revealed several important features related to exosite binding that provide a structural rationale for the high specificity and affinity of VEK-30 for K2(Pg)...
  21. pmc Plasminogen enhances neuritogenesis on laminin-1
    Ana Gutierrez-Fernandez
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    J Neurosci 29:12393-400. 2009
    ..This study provides the first direct evidence that plasminogen participates in neurite outgrowth and also suggests that laminin-1 degradation by plasmin contributes to the process of neuritogenesis...
  22. ncbi Plasminogen receptors: the sine qua non of cell surface plasminogen activation
    Lindsey A Miles
    Department of Cell Biology, Division of Vascular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    Front Biosci 10:1754-62. 2005
    ..This issue includes chapters on the well studied plasminogen receptor functions...